PROJECT SUMMARY We propose to develop and validate an intracellular stained Fluorescence Activated Cell Sorter Templated Oligonucleotide Sequencing gene profiling assay, icsFACS/TempO-Seq, addressing an unmet need by enabling low-cost, quantitative gene expression analysis of fixed, permeabilized, and intracellular stained and sorted cells (a method used to purify many different functionally important and rare subsets of cells), including single cells, on both the FACSAria and benchtop BD FACSMelody, Sony SH800, and BioRad S3e sorters. We successfully demonstrated feasibility in Phase I, developing two protocols for profiling single FACS sorted cells. In addition, we were able to demonstrate novel observations regarding the stochastic expression of genes at the single cell level due to the performance of the assay and virtual absence of background signal so that true ?0? expression levels of genes that were off in single cells at the time of fixation could be measured. In Phase II we will optimize these protocols and validate a menu of assays for human and mouse ? a whole transcriptome assay, a surrogate assay, a panel comprised of the surrogate assay plus immune response genes for both mouse and human, and a panel comprised of host response genes, HIV-1 viral RNA and DNA. We will demonstrate utility to generate time course data of normal T-cells activated ex vivo for subsets and single cells within those subsets. Finally, we will generate novel data in the course of demonstrating utility in a mouse model carried out in collaboration with an expert consultant, and by data obtained from HIV-1 patients in a ?-test client arrangement with an expert in HIV research. In the mouse model we will profile GC B cells undergoing programmed rounds of hypermutation and selection (proliferation) and monitor subset purity in a notoriously coalescing surface-staining phenotype. In the human model, the ?-test client will profile FACS sorted samples from HIV-1 infected patients undergoing triple antiretroviral therapy (ART) to characterize the host RNA response and HIV-1 provirus DNA and viral mRNA harbored within the CD4+ T memory cell compartments and enumerate residual HIV+ CD4+ T cells. The assay used will include measurement of viral RNA and DNA at the same time as host genes. The icsFACS/TempO-Seq protocols and assays developed and validated will address unmet needs in flow cytometry and immunology across a growing range of diseases, including infectious diseases, inflammation, cancer, stroke, and Alzheimer?s to name a few. Validation for both the FACSAria and benchtop sorters will address the needs of core labs and their clients and investigators who choose to use, or require the specialized single cell performance, of these benchtop sorters. It also lays the groundwork for the development of diagnostic assays on these benchtop sorter platforms using icsFACS/TempO-Seq.